Unique model of chronic hypoxia in fetal lambs demonstrates abnormal contrast-enhanced ultrasound brain perfusion.

BACKGROUND: Children with congenital heart disease (CHD) demonstrate long-term neurodevelopmental impairments. We investigated contrast-enhanced ultrasound (CEUS) cerebral perfusion in a fetal animal model exposed to sub-physiologic oxygen at equivalent levels observed in human fetuses with CHD. METHODS: Fifteen fetal lambs [hypoxic animals (n = 9) and normoxic controls (n = 6)] maintained in an extrauterine environment underwent periodic brain CEUS. Perfusion parameters including microvascular flow velocity (MFV), transit time, and microvascular blood flow (MBF) were extrapolated from a standardized plane; regions of interest (ROI) included whole brain, central/thalami, and peripheral parenchymal analyses. Daily echocardiographic parameters and middle cerebral artery (MCA) pulsatility indices (PIs) were obtained. RESULTS: Hypoxic lambs demonstrated decreased MFV, increased transit time, and decreased MBF (p = 0.026, p = 0.016, and p < 0.001, respectively) by whole brain analyses. MFV and transit time were relatively preserved in the central/thalami (p = 0.11, p = 0.08, p = 0.012, respectively) with differences in the peripheral parenchyma (all p < 0.001). In general, cardiac variables did not correlate with cerebral CEUS perfusion parameters. Hypoxic animals demonstrated decreased MCA PI compared to controls (0.65 vs. 0.78, respectively; p = 0.027). CONCLUSION: Aberrations in CEUS perfusion parameters suggest that in environments of prolonged hypoxia, there are regional microvascular differences incompletely characterized by MCA interrogation offering insights into fetal conditions which may contribute to patient outcomes. IMPACT: This work utilizes CEUS to study cerebral microvascular perfusion in a unique fetal animal model subjected to chronic hypoxic conditions equal to fetuses with congenital heart disease. CEUS demonstrates altered parameters with regional differences that are incompletely characterized by MCA Doppler values. These findings show that routine MCA Doppler interrogation may be inadequate in assessing microvascular perfusion differences. To our knowledge, this study is the first to utilize CEUS to assess microvascular perfusion in this model. The results offer insight into underlying conditions and physiological changes which may contribute to known neurodevelopmental impairments in those with congenital heart disease.

Assessment of extremely premature lambs supported by the Extrauterine Environment for Neonatal Development (EXTEND).

BACKGROUND: Our team has previously reported physiologic support by the EXTra-uterine Environment for Neonatal Development (EXTEND) of 105 to 117 days gestational age (GA) lambs for up to 28 days with normal organ maturation. However, the fetal lamb brain matures more rapidly, requiring the study of 90-105 day GA fetal lambs to assess more neurodevelopmentally equivalent lambs to the 23-25 week GA extreme premature infant. METHODS: Extremely preterm lambs (90-95 days of GA) were delivered by C-section and supported by EXTEND. Estimated circuit flows were maintained at around 325 ml/kg/min. After support on EXTEND, MRI and histopathologic analysis were performed and compared to 105-112 days GA control lambs. RESULTS: The extremely preterm group includes 10 animals with a mean GA of 91.6 days, a mean weight at cannulation of 0.98 kg and a mean length of stay on EXTEND of 13.5 days (10-21 days). Hemodynamics and oxygenation showed stable parameters. Animals showed growth and physiologic cardiac function. MRI volumetric and diffusion analysis was comparable to controls. Histologic brain analysis revealed no difference between study groups. CONCLUSION: EXTEND appears to support brain and cardiac development in an earlier gestation, less mature, lamb model. IMPACT: Prolonged (up to 21 days) physiological support of extremely preterm lambs of closer neurodevelopmental equivalence to the 24-28 gestational week human was achieved using the EXTEND system. EXTEND treatment supported brain growth and development in extremely preterm fetal lambs and was not associated with intraventricular hemorrhage or white matter injury. Daily echocardiography demonstrated physiologic heart function, absence of cardiac afterload, and normal developmental increase in cardiac chamber dimensions. This study demonstrates hemodynamic and metabolic support by the EXTEND system in the extremely preterm ovine model.

A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

Radiographic and histologic characterisation of white matter injury in a sheep model of CHD.

Nearly one in five children with CHD is born with white matter injury that can be recognised on postnatal MRI by the presence of T1 hyperintense lesions. This pattern of white matter injury is known to portend poor neurodevelopmental outcomes, but the exact aetiology and histologic characterisation of these lesions have never been described. A fetal sheep was cannulated at gestational age 110 days onto a pumpless extracorporeal oxygenator via the umbilical vessels and supported in a fluid environment for 14.5 days. The fetus was supported under hypoxic conditions (mean oxygen delivery 16 ml/kg/day) to simulate the in utero conditions of CHD. At necropsy, the brain was fixed, imaged with MRI, and then stained to histologically identify areas of injury. Under hypoxemic in utero conditions, the fetus developed a T1 hyperintense lesion in its right frontal lobe. Histologically, this lesion was characterised by microvascular proliferation and astrocytosis without gliosis. These findings may provide valuable insight into the aetiology of white matter injury in neonates with CHD.

The EXTrauterine Environment for Neonatal Development: Present and Future.

Despite advances in clinical care and modest improvement in mortality rates for extreme prematurity, morbidity remains a significant challenge. The ideal environment to support prematurity would be fluidic and rely on natural fetal circulation to mimic the natural fetal amniotic environment, yet such an environment has been unsuccessful in long-term support until recently. Our group has succeeded in developing such a support system to foster fetal growth in the premature lamb model that shows promise for clinical translation. Here, we describe the EXTrauterine Environment for Neonatal Development (EXTEND) from its conception onwards, review published literature on fetal development and support of the premature lamb model in EXTEND, and discuss future applications.

Artificial placenta and womb technology: Past, current, and future challenges towards clinical translation.

Extreme prematurity remains a major cause of neonatal mortality and severe long-term morbidity. Current neonatal care is associated with significant morbidity due to iatrogenic injury and developmental immaturity of extreme premature infants. A more physiologic approach, replacing placental function and providing a womb-like environment, is the foundational principle of artificial placenta (AP) and womb (AW) technology. The concept has been studied during the past 60 years with limited success. However, recent technological advancements and a greater emphasis on mimicking utero-placental physiology have improved the success of experimental models, bringing the technology closer to clinical translation. Here, we review the rationale for and history of AP and AW technology, discuss the challenges that needed to be overcome, and compare recent successful models. We conclude by outlining some remaining challenges to be addressed on the path towards clinical translation and opportunities for future research.

Evaluation of umbilical venous flow volume measured using ultrasound compared to circuit flow volume in the EXTra-uterine Environment for Neonatal Development (EXTEND) system in fetal sheep.

OBJECTIVE: To compare and validate umbilical venous flow volume (UVFV) measured at the intra-abdominal portion using ultrasound with actual flow volume of umbilical vein (UV) in fetal sheep sustained on the EXTrauterine Environment for Neonatal Development (EXTEND) system. METHODS: Circuit flow volume through the oxygenator was obtained using sensors. Ultrasound derived UVFV (ml/min) was calculated as (UV diameter [cm]/2)2 × 3.14 × maximum velocity (cm/s) × 0.5 × 60, measured at approximately the mid portion between its abdominal insertion and the origin of the ductus venosus. UVFV was measured by ultrasound once daily and was compared to the average of daily circuit flow volume directly measured. RESULTS: UVFV was measured 168 times in 15 fetal sheep. The ratio of circuit flow volume to combined cardiac output remained stable within the anticipated physiological range throughout. UVFV measured by ultrasound showed good correlation to directly measured circuit flow (r = 0.72). Interclass correlation coefficients for intra-observer variability was 0.991 (95% confidence interval [CI], 0.979-0.996). CONCLUSION: UVFV measured at the intra-abdominal portion using ultrasound shows a good correlation with directly measured circuit flow volume in UV of fetal sheep on the EXTEND system. Regular incorporation of such validated UVFV measures into clinical use may offer opportunities to better understand conditions of placental dysfunction.

Contrast-Enhanced Brain Ultrasound Perfusion Metrics in the EXTra-Uterine Environment for Neonatal Development (EXTEND): Correlation With Hemodynamic Parameters.

OBJECTIVES: Contrast-enhanced ultrasound (CEUS) can provide quantitative perfusion metrics and may be useful to detect cerebral pathology in neonates and premature infants, particularly in extrauterine environments. The effect of hemodynamics on cerebral perfusion metrics is unknown, which limits the clinical application of this technology. We aimed to determine associations between systemic hemodynamics and concurrently measured brain perfusion parameters in an animal model of extrauterine support. METHODS: Nine fetal lambs were transferred to an extrauterine support device. Lumason® ultrasound contrast (0.1-0.3 ml) was administered via the umbilical vein and 90-second cine clips were obtained. Time-intensity-curves (TICs) were generated and time-dependent and area-under-curve (AUC) parameters were derived. Associations between brain perfusion metrics and hemodynamics including heart rate (HR) and mean arterial pressure (MAP) were evaluated by multilevel linear mixed-effects models. RESULTS: Eighty-six ultrasound examinations were performed and 72 examinations were quantifiable. Time-dependent measurements were independent of all hemodynamic parameters (all p ≥.05). Oxygen delivery and mean blood flow were correlated with AUC measurements (all p ≤.01). Physiologic HR and MAP were not correlated with any measurements (all p ≥.05). CONCLUSION: Detected aberrations in time-dependent CEUS measurements are not correlated with hemodynamic parameters and are thought to reflect the changes in cerebral blood flow, thus providing a promising tool for evaluation of brain perfusion. CEUS brain perfusion parameters are not correlated with physiologic HR and MAP, but AUC-dependent measurements are correlated with oxygen delivery and blood flow, suggesting that CEUS offers additional value over standard monitoring. Overall, these findings enhance the applicability of this technology.

Quantitative contrast-enhanced ultrasound of the brain on twin fetal lambs maintained by the extrauterine environment for neonatal development (EXTEND): initial experience.

BACKGROUND: With the development of an artificial environment to support the extremely premature infant, advanced imaging techniques tested in this extrauterine system might be beneficial to evaluate the fetal brain. OBJECTIVE: We evaluated the feasibility of (a) performing contrast-enhanced ultrasound (CEUS) and (b) quantifying normal and decreased brain perfusion in fetal lambs maintained on the extrauterine environment for neonatal development (EXTEND) system. MATERIALS AND METHODS: Twin premature fetal lambs (102 days of gestational age) were transferred to the EXTEND system. Twin B was subjected to sub-physiological flows (152 mL/kg/min) and oxygen delivery (15.9 mL/kg/min), while Twin A was maintained at physiological levels. We administered Lumason contrast agent into the oxygenator circuit and performed serial CEUS examinations. We quantified perfusion parameters and generated parametric maps. We also recorded hemodynamic parameters, serum blood analysis, and measurements across the oxygenator. Postmortem MRIs were performed. RESULTS: No significant changes in hemodynamic variables were attributable to CEUS examinations. On gray-scale images, Twin B demonstrated ventriculomegaly and progressive parenchymal volume loss culminating in hydranencephaly. By CEUS, Twin B demonstrated decreased peak enhancement and decreased overall parenchymal perfusion when compared to Twin A by perfusion parameters and parametric maps. Changes in perfusion parameters were detected immediately following blood transfusion. Postmortem MRI confirmed ultrasonographic findings in Twin B. CONCLUSION: In this preliminary experience, we show that CEUS of the brain is feasible in fetal lambs maintained on the EXTEND system and that changes in perfusion can be quantified, which is promising for the application of CEUS in this extrauterine system supporting the premature infant.

Technical feasibility of umbilical cannulation in midgestation lambs supported by the EXTra-uterine Environment for Neonatal Development (EXTEND).

EXTEND (EXTra-uterine Environment for Neonatal Development) is a novel system for supporting extremely premature infants that replicates in utero conditions by maintaining a sterile fluid environment and providing gas exchange via a pumpless arteriovenous oxygenator circuit connected to the umbilical vessels. Target gestational age (GA) for EXTEND support in human infants is 23-27 weeks, when immature lungs are most susceptible to injury in the setting of air ventilation. We previously demonstrated physiologic support of premature lambs cannulated at 105-117 days GA (lungs developmentally analogous to the 23-27 week GA human infant) for up to 28 days on EXTEND. In the present study, we sought to determine the technical feasibility of umbilical vessel cannulation in 85-96 days GA lambs delivered to EXTEND at weights equivalent to the 23-27 week GA human infant (500-850 g). Five preterm lambs were cannulated at 85-96 days GA (term 145 days) and supported on EXTEND for 4-7 days. All lambs underwent umbilical artery and umbilical vein cannulation. Circuit flows and pressures were monitored continuously, and blood gases were obtained at regular intervals for assessment of oxygen parameters. Systemic pH and lactate were measured at least once daily. Mean body weight at cannulation was 641 ± 71 g (range 480-850 g). All lambs were cannulated successfully (cannula size varied from 8 to 12 Fr), and mean survival on EXTEND was 140 ± 7 hours. Mean circuit flow was 213 ± 15 mL/kg*min, mean pH was 7.37 ± 0.01, and mean lactate was 1.6 ± 0.2 mmol/L. During the initial 120 hours after EXTEND cannulation, there were no significant differences between 85-96 days GA lambs and 105-117 days GA lambs in weight-adjusted circuit flows, oxygen delivery, pH, or lactate levels. This study demonstrates successful umbilical cord cannulation and adequate circuit flows and oxygen delivery in midgestation lambs size-matched to the 23-27 week GA human fetus, which represents an important step in the translation of EXTEND to clinical practice.

Contrast-Enhanced Ultrasound in Extracorporeal Support: In Vitro Studies and Initial Experience and Safety Data in the Extreme Premature Fetal Lamb Maintained by the Extrauterine Environment for Neonatal Development.

OBJECTIVES: To evaluate the effects of ultrasound contrast agent (UCA) administration on hemodynamic parameters and support equipment in in vitro and in vivo models of extracorporeal support. METHODS: In vitro, incrementally increasing bolus doses of a UCA were administered proximal to a membrane oxygenator, and ultrasound cine clips were obtained. The rates of microbubble destruction across the oxygenator and over time were calculated from time-intensity-curves. Measurements across the membrane oxygenator were recorded and compared by a repeated-measures analysis of variance. In vivo, 7 premature fetal lambs were transferred from placental support to the extrauterine environment for neonatal development. Contrast agent boluses were administered for contrast-enhanced ultrasound (CEUS) examinations. Hemodynamic parameters and serum laboratory values were evaluated before and after the examinations by paired t tests. For oxygenator staining, oxygenator membranes from the in vitro circuit, study animals (n = 4), and control animals (n = 4) were stained for the adherent UCA. RESULTS: In vitro, with all doses (0.1-4 mL), there was no difference in measured parameters across the oxygenator (P ≥ .09). Contrast agent destruction (3%-14%) across the oxygenator was observed at the first pass with a progressive decline in contrast intensity over time. In vivo, there was no difference in hemodynamic parameters or serum laboratory values (P ≥ .08) with any CEUS examination (n = 17). For oxygenator staining, all oxygenator membranes were negative for UCA with lipid staining. CONCLUSIONS: The UCA had no detectable effect on the oxygenator or measured parameters in in vitro and in vivo studies, thus providing additional safety data to support the use of CEUS in the setting of extracorporeal support.

Chronically Hypoxic Fetal Lambs Supported by an Extra-Uterine Device Exhibit Mitochondrial Dysfunction and Elevations of Hypoxia Inducible Factor 1-Alpha.

INTRODUCTION: We have recently developed an extra-uterine environment for neonatal development (EXTEND) capable of supporting premature fetal lambs and have been able to replicate hypoxic in utero conditions by controlling fetal oxygen delivery. In this study, we investigated the fetal mitochondrial response to hypoxia. METHODS: Eight premature fetal lambs were delivered via hysterotomy and transitioned to extra-uterine support for 3 weeks. The lambs were divided into 2 groups: normoxic fetuses which maintained physiologic oxygen delivery and hypoxic fetuses in which oxygen delivery was significantly reduced. Control fetuses were delivered via hysterotomy but not cannulated. Measurements of mitochondrial membrane potential (MMP) were performed in peripheral blood mononuclear cells. RESULTS: There were no significant differences in MMP between normoxic EXTEND fetuses and controls. Hypoxic fetuses had significantly more depolarized mitochondria compared to normoxic fetuses overall, and these changes were specifically appreciated in weeks 1 and 2, but not by week 3. Hypoxic fetuses had significantly elevated levels of HIF-1α compared to normoxic fetuses in the first 2 weeks. DISCUSSION: Normoxic fetal lambs supported by EXTEND demonstrate normal mitochondrial function as evidenced by equivalent membrane potentials compared to control fetuses. Hypoxic fetuses exhibit mitochondrial dysfunction, though they do show evidence of adaptation after 3 weeks of hypoxic exposure.

Premature Lambs Exhibit Normal Mitochondrial Respiration after Long-Term Extrauterine Support.

BACKGROUND: In an effort to mitigate the major morbidities and mortality associated with extreme prematurity, we have developed an EXTrauterine Environment for Neonatal Development (EXTEND) designed to provide physiologic support of extremely premature infants. OBJECTIVES: We have previously shown that long-term, physiologic support of premature fetal lambs is possible with EXTEND, but in this study, we sought to demonstrate bioenergetic equipoise at the tissue level. METHODS: Four premature fetal lambs were delivered by hysterotomy at gestational ages (GA) of 105-107 days (term ∼145 days), cannulated via the umbilical vessels, and transitioned to support on EXTEND for 3-4 weeks. Five control fetuses were age-matched to the GA of experimental fetuses at the time of study end (128-134 days GA) and immediately sacrificed after hysterotomy. Mitochondria were isolated from the heart, liver, kidney, and skeletal muscle of fetuses at the time of sacrifice, and oxygen consumption rates (OCRs) were measured. RESULTS: There were no differences in basal mitochondrial OCR between EXTEND and control fetuses for heart, kidney, or skeletal muscle. For liver, the basal OCR was higher in EXTEND fetuses compared to controls. There were no differences in physiologic maximal OCR or reserve capacity for any tissue analyzed. CONCLUSIONS: Fetal lambs supported by EXTEND demonstrate physiologic mitochondrial function as evidenced by adequate basal and physiologic maximal cellular respiration as well as preserved reserve capacity.

Erythropoietin Prevents Anemia and Transfusions in Extremely Premature Lambs Supported by an EXTrauterine Environment for Neonatal Development (EXTEND).

BACKGROUND: We recently developed an EXTrauterine Environment for Neonatal Development (EXTEND) that provides physiologic support for premature lambs. Here, we assess the efficacy of exogenous erythropoietin (EPO) to prevent anemia and transfusions on EXTEND. MATERIALS AND METHODS: Lambs were cannulated at 0.7 gestation and supported on EXTEND for up to 4 weeks. The lambs were divided into three groups: (1) No EPO, (2) Low EPO (300 U kg-1 per day), and (3) High EPO (800 U kg-1 per day). Daily hematocrit and weekly complete blood count were assessed. RESULTS: The mean percentage change in hematocrit from baseline was significantly different between the groups (No EPO -23.6 ± 7.8% vs. Low EPO -16.6 ± 6.4% vs. High EPO +2.6 ± 6.6%; p = 0.02). This occurred despite a greater median number of blood transfusions in the No EPO group (5 vs. 1 vs. 0; p = 0.02). EPO administration was associated with a higher mean corpuscular volume (MCV; p < 0.01) and reticulocyte count (p = 0.02). The High EPO group was comparable to in utero control fetuses with respect to hematocrit (p = 0.49), MCV (p = 0.24), and reticulocyte count (p = 0.68). CONCLUSIONS: EPO (800 U kg-1 per day) prevents anemia, eliminates transfusions, and restores normal red blood cell indices in premature lambs supported by EXTEND.

Umbilical cannulation optimizes circuit flows in premature lambs supported by the EXTra-uterine Environment for Neonatal Development (EXTEND).

KEY POINTS: Bronchopulmonary dysplasia is a disease of extreme prematurity that occurs when the immature lung is exposed to gas ventilation. We designed a novel 'artificial womb' system for supporting extreme premature lambs (called EXTEND) that obviates gas ventilation by providing oxygen via a pumpless arteriovenous circuit with the lamb submerged in sterile artificial amniotic fluid. In the present study, we compare different arteriovenous cannulation strategies on EXTEND, including carotid artery/jugular vein (CA/JV), carotid artery/umbilical vein (CA/UV) and umbilical artery/umbilical vein (UA/UV). Compared to CA/JV and CA/UV cannulation, UA/UV cannulation provided significantly higher, physiological blood flows to the oxygenator, minimized flow interruptions and supported significantly longer circuit runs (up to 4 weeks). Physiological circuit blood flow in UA/UV lambs made possible normal levels of oxygen delivery, which is a critical step toward the clinical application of artificial womb technology. ABSTRACT: EXTEND (EXTra-uterine Environment for Neonatal Development) is a novel system that promotes physiological development by maintaining the premature lamb in a sterile fluid environment and providing gas exchange via a pumpless arteriovenous oxygenator circuit. During the development of EXTEND, different cannulation strategies evolved with the aim of improving circuit flow. The present study examines how different cannulation strategies affect EXTEND circuit haemodynamics in extreme premature lambs. Seventeen premature lambs were cannulated at gestational ages 105-117 days (term 145-150 days) and supported on EXTEND for up to 4 weeks. Experimental groups were distinguished by cannulation strategy: carotid artery outflow and jugular vein inflow (CA/JV; n = 4), carotid artery outflow and umbilical vein inflow (CA/UV; n = 5) and double umbilical artery outflow and umbilical vein inflow (UA/UV; n = 8). Circuit flows and pressures were measured continuously. As we transitioned from CA/JV to CA/UV to UA/UV cannulation, mean duration of circuit run and weight-adjusted circuit flows increased (P < 0.001) and the frequency of flow interruptions declined (P < 0.05). Umbilical vessels generally accommodated larger-bore cannulas, and cannula calibre was directly correlated with circuit pressures and indirectly correlated with flow:pressure ratio (a measure of post-membrane resistance). We conclude that UA/UV cannulation in fetal lambs on EXTEND optimizes circuit flow dynamics and flow stability and also supports circuit flows that closely approximate normal placental flow.

A longer tracheal occlusion period results in increased lung growth in the nitrofen rat model.

OBJECTIVE: Prenatal tracheal occlusion (TO) promotes lung growth and is applied clinically in fetuses with severe congenital diaphragmatic hernia. Limited data are available regarding the effect of duration of TO on lung development. Our objective was to evaluate the effects of long (2 and 2.5 days) versus short (1 day) TO on lung development in rats with nitrofen-induced diaphragmatic hernia. METHOD: Nitrofen was administered on embryonic day (ED) 9 and fetal TO performed either on ED18.5, 19 or 20 (term = 22 days). Sham-operated and untouched littermates served as controls. On ED21, lungs were harvested and only fetuses with a left-sided diaphragmatic defect were included in further analyses. RESULTS: Lung-body-weight ratio incrementally increased with the duration of TO. Increased proliferation following long TO was confirmed by immunohistochemistry and qRT-PCR for the proliferation marker Ki-67. Irrespective of duration, TO induced more complex airway architecture. Medial wall thickness of pulmonary arteries was thinner after long rather than short TO. CONCLUSION: In the nitrofen rat model of congenital diaphragmatic hernia, a longer period of TO leads to enhanced lung growth and less muscularized pulmonary arteries.

Chronic hypoxemia induces mitochondrial respiratory complex gene expression in the fetal sheep brain.

Objective: The molecular pathways underlying hypoxemia-induced alterations in neurodevelopment of infants with congenital heart disease have not been delineated. We used transcriptome analysis to investigate differential gene expression induced by hypoxemia in an ovine artificial-womb model. Methods: Mid-gestation fetal sheep (median [interquartile range] 109 [107-112] days' gestation) were cannulated via the umbilical vessels, attached to a pumpless, low-resistance oxygenator circuit, and incubated in a sterile, fluid environment for 22 [21-23] days. Fetuses were maintained with an oxygen delivery of 20-25 mL/kg/min (normoxemia, n = 3) or 14-16 mL/kg/min (hypoxemia, n = 4). Transcriptional profiling by RNA sequencing was carried out on left frontal brains and hypoxemia-regulated genes were identified by differential gene expression analysis. Results: A total of 228 genes whose expression was up or down regulated by ≥1.5-fold (false discovery rate ≤0.05) were identified. The majority of these genes were induced in hypoxemic animals compared to normoxemic controls, and functional enrichment analysis identified respiratory electron transport as a pathway strongly upregulated in the brain during chronic hypoxemia. Further examination of hypoxemia-induced genes showed robust induction of all 7 subunits of the mitochondrial NADH:ubiquinone oxidoreductase (complex I). Other hypoxemia-induced genes included cytochrome B, a component of complex III, and ATP6, ATP8, both of which are components of complex V. Conclusions: Chronic fetal hypoxemia leads to upregulation of multiple mitochondrial respiratory complex genes critical for energy production and reactive oxygen species generation, including complex I. These data provide valuable insight into potential pathways involved in chronic hypoxemia-induced neuropathology and offers potential therapeutic targets for fetal neuroprotection in fetuses with congenital heart defects.

Pharmacokinetics and pharmacodynamics of sildenafil in fetal lambs on extracorporeal support.

BACKGROUND: Maternal transplacental administration of sildenafil is being considered for a variety of fetal conditions. Clinical translation also requires evaluation of fetal safety in a higher species, such as the fetal lamb. Experiments with the pregnant ewe are curtailed by minimal transplacental transfer as well as limited access to the fetus. The EXTra-uterine Environment for Neonatal Development (EXTEND) model renders the isolated fetal lamb readily accessible and allows for direct fetal administration of sildenafil. METHODS: Five fetal lambs were placed on extracorporeal support in the EXTEND device and received continuous intravenous (IV) sildenafil (0.3-0.5-0.7 mg/kg/24hr) for a duration of one to seven days. Plasma sildenafil concentrations were sampled at regular intervals to establish the pharmacokinetic profile using population pharmacokinetic modeling. Serial Doppler ultrasound examination, continuous non-invasive hemodynamic monitoring and blood gas analysis were done to evaluate the pharmacodynamic effects and fetal response. FINDINGS: The target concentration range (47-500 ng/mL) was attained with all doses. Sildenafil induced an immediate and temporary reduction of pulmonary vascular resistance, mean arterial pressure and circuit flow, without change in fetal lactate levels and acid-base status. The duration of the systemic effects increased with the dose. INTERPRETATION: Immediate temporary pulmonary vascular and systemic hemodynamic changes induced by sildenafil were biochemically well tolerated by fetal lambs on extracorporeal support, with the 0.5 mg/kg/24 h dose balancing rapid attainment of target concentrations with short-lived systemic effects. RESEARCH IN CONTEXT: None. SEARCH STRATEGY BEFORE UNDERTAKING THE STUDY: A literature review was conducted searching online databases (Medline, Embase and Cochrane), using search terms: fetal OR prenatal OR antenatal AND sildenafil, without time-limit and excluding human studies. Where relevant, investigators were contacted in order to avoid duplication of work. EVIDENCE BEFORE THIS STUDY: Prenatal therapy with sildenafil, a phosphodiesterase-5 inhibitor with vasodilatory and anti-remodeling effects on vascular smooth muscle cells, has been considered for a variety of fetal conditions. One multicenter clinical trial investigating the benefit of sildenafil in severe intrauterine growth restriction (the STRIDER-trial) was halted early due to excess mortality in the sildenafil-exposed arm at one treatment site. Such findings demonstrate the importance of extensive preclinical safety assessment in relevant animal models. Transplacentally administered sildenafil leads to decreased pulmonary arterial muscularization, preventing or reducing the occurrence of pulmonary hypertension in rat and rabbit fetuses with diaphragmatic hernia (DH). Validation of these results in a higher and relevant animal model, e.g. fetal lambs, is the next step to advance clinical translation. We recently demonstrated that, in contrast to humans, transplacental transfer of sildenafil in sheep is minimal, precluding the in vivo study of fetal effects at target concentrations using the conventional pregnant ewe model. ADDED VALUE OF THIS STUDY: We therefore used the extracorporeal support model for fetal lambs, referred to as the EXTra-uterine Environment for Neonatal Development (EXTEND) system, bypassing placental and maternal metabolism, to investigate at what dose the target concentrations are reached, and what the fetal hemodynamic impact and response are. Fetal hemodynamic and metabolic tolerance to sildenafil are a crucial missing element on the road to clinical translation. This is therefore the first study investigating the pharmacokinetics, hemodynamic and biochemical effects of clinical-range concentrations of sildenafil in fetal lambs, free from placental and maternal interference. IMPLICATIONS OF ALL THE AVAILABLE EVIDENCE: We demonstrated self-limiting pulmonary vasodilation, a decrease of both systemic arterial pressures and circuit flows, induced by clinical range concentrations of sildenafil, without the development of fetal acidosis. This paves the way for further investigation of prenatal sildenafil in fetal lambs on extracorporeal support. A dose of 0.5 mg/kg/24 h offered the best trade-off between rapid achievement of target concentrations and shortest duration of systemic effects. This is also the first study using the EXTEND as a model for pharmacotherapy during pregnancy.

Fetal tendon wound size modulates wound gene expression and subsequent wound phenotype.

The fetal response to small tendon injury results in regenerative or scarless healing and is characterized by a markedly diminished cellular inflammatory response, lack of fibroplasia, and restoration of normal tissue architecture. We hypothesized that an increasing fetal tendon wound size would lead to increased wound inflammation and a change from regenerative to reparative healing and scar formation. We created small or large tendon wounds in early gestation fetal sheep and used histology to assess tissue architecture, immunohistochemistry to assess the cellular inflammatory response, ovine-specific gene microarrays, and real-time reverse transcription-polymerase chain reaction to measure the gene expression in response to injury. Small tendon wounds showed a regenerative healing phenotype with orderly deposition of collagen fibers while large tendon wounds showed disorderly collagen deposition consistent with scar formation. Small tendon wounds had few inflammatory cells at 7 and 28 days after injury, whereas the large wounds showed a significant inflammatory cell infiltrate at 7 days that resolved by 28 days. At 3 days, the differential expression of genes involved in the response to injury and inflammation were seen between large and small tendon wounds. By real-time polymerase chain reaction at 7 days, large tendon wounds also had significantly increased expression of interleukin-6, interleukin-8, transforming growth factor-ß1, and transforming growth factor-ß3, compared with the small wounds. Increasing the fetal tendon wound size results in increased proinflammatory gene expression, inflammatory cell infiltration, and a change from regenerative to reparative healing. This model allows the process of regenerative healing to be examined without the confounding variable of gestational age.

The Effects of Nitric Oxide in Oxygenator Sweep Gas During Extracorporeal Circulation in a Neonatal Ovine Model.

Extracorporeal membrane oxygenation is a life-saving intervention, but bleeding complications are frequent. Given that the combination of platelet loss and dysfunction is a major contributor to this acquired bleeding diathesis, efforts to combat these phenomena are of great clinical importance. In this study, we investigated the effects of nitric oxide (NO) added to the sweep gas of an extracorporeal circuit in a neonatal ovine model. Eight lambs (age 9.6 ± 1.9 days) were cannulated via the neck vessels and maintained on a pumpless arteriovenous extracorporeal membrane oxygenation circuit with blood flow restricted to 100 ml/min for 72 hours. All animals were heparinized, and a subset (n = 4) also received NO in the sweep gas at a concentration of 200 ppm. We observed no adverse effects from NO administration, and methemoglobin levels remained unchanged. Platelet counts significantly declined in all animals over the course of the study; however, mean counts were higher in the NO-treated group, and this difference was statistically significant at 24 hours (62 ± 3% vs. 32 ± 7% of baseline, P < 0.01). Likewise, mean plasma levels of beta-thromboglobulin, a marker of platelet activation, were lower in the NO-treated group, and this difference was also significant at the 24 hour time point (9.5 ± 2.2 vs. 19.7 ± 6.5 pg/mL/10 platelets, P < 0.05). We conclude that 200 ppm NO can be safely blended into the oxygenator sweep gas of a low-flow extracorporeal circuit and that it may transiently attenuate platelet consumption and activation.